The Study Of The Contents And The Role Of Platelet-rich Fibrin In Dental Pulp Cells Regeneration

Part 1: Effect of liquid-PRF and PRP on the regenerative potential of dental pulp cells cultured under inflammatory conditions: a comparative analysis.Objectives: Platelet-rich plasma(PRP)has been widely used in regenerative dentistry for over two decades.Nevertheless,previous studies have shown that the application of anti-coagulants limits its regenerative potential.While liquid platelet rich fibrin(liquid-PRF)was developed without the use of anti-coagulants and shorter centrifugation times.The purpose of the present study was to compare the biological activities of liquid-PRF with traditional PRP on human dental pulp cells(h DPCs)in vitro.The regenerative potential of h DPCs was investigated in both normal and inflammatory-like conditions(LPS)and assessed for their potential for dentin repair.Materials and methods: The effects of liquid-PRF and PRP was assessed for cellular migration,proliferation,and odontoblastic differentiation using a transwell assay,scratch assay,proliferation assay,alkaline phosphatase(ALP)assay,alizarin red staining,and real-time PCR for genes encoding collagen1a1,DSPP,and DMP-1,respectively.The effects of both platelet concentrates were also assessed for their ability to influence nuclear translocation of NF-?B(p65)by immunofluorescence,and RT-PCR for genes encoding interleukin-1?(IL-1?),tumor necrosis factor-?(TNF-?),and nuclear factor kappa B(p65)during an inflammatory condition.Results: The results demonstrated that both PRP and liquid-PRF increased the migration and proliferation of h DPCs when compared to the control group,while liquid-PRF demonstrated a notable significant increase in migration when compared to PRP.Furthermore,liquid-PRF induced significantly greater ALP activity,alizarin red staining and a m RNA expression of gene encoding Col1a1,DSPP and DMP-1 when compared to PRP.When h DPCs were cultured with LPS to stimulate an inflammatory environment,a marked decrease in dentin-related repair was observed.When liquid-PRF was cultured within this inflammatory environment,the reduced regenerative potential in this LPS-produced environment was significantly and markedly improved facilitating h DPC regeneration.The m RNA expression of inflammatory markers including TNF-?,IL-1?,and p65 were all significantly decreased in the presence of liquid-PRF and furthermore,liquid-PRF also inhibited the transport of p65 to the nucleus in h DPCs(suggesting a reduced inflammatory condition).Conclusions: liquid-PRF promoted significantly greater regeneration potential of h DPCs when compared to traditional PRP.Furthermore,liquid-PRF also attenuated the inflammatory condition created by LPS and maintained a supportive regenerative ability for the stimulation of odontoblastic differentiation and reparative dentin in h DPCs.Part 2: Evaluating and Quantifying Cell Types in Platelet Rich FibrinObjectives: Platelet rich fibrin(PRF)has been utilized clinically as a platelet concentrate capable of stimulating tissue regeneration.Interestingly,several protocols have been proposed with little data obtained regarding the final cell counts.The aim of the present study was to compare different commercially available centrifuges and their respective protocols utilizing a novel method to quantify cells in the various blood layers.Materials and Methods: PRF was produced on three centrifuges including 2 fixed-angle centrifuges(Intra Spin(Intra Lock)and The Duo Quattro)and Eppendorf.Two separate protocols were tested on each device including protocols to produce either liquid-PRF and solid-PRF(Intraspin = ~700g for 3 and 12 minutes respectively;The Duo Quattro = ~60g for 3 minutes and ~200g for 8 minutes;and 200 g and 700 g for 8 minutes on a Eppendorf centrifuge).Following centrifugation,1m L layers were sequentially pipetted from the upper layer of blood tubes towards the bottom of the tube until all 10 m L were harvested in sequential samples.10 samples from each blood draw was then sent for complete blood count(CBC)analysis to accurately quantify precisely cell numbers within each separate blood layer.Results: The present study revealed marked differences in cell numbers within the various samples.L-PRF protocols(Intra Spin;700g)produced a clot with the majority of platelets and leukocytes concentrated within the buffy coat(layer precisely above the red blood corpuscle layer)with relatively no cells found within the first 4 m L of L-PRF.Slower centrifugation protocols produced using the A-PRF protocols(The Duo Quattro;200g)produced a more evenly distributed number of platelets throughout the PRF layer,however a significantly lower number and concentration of leukocytes.The injectable-PRF(i-PRF)protocol(60g for 3 minutes)produced the highest concentration of leukocytes and platelets when compared to either Intra Spin protocols,however the total number of leukocytes and platelets were significantly lower owing to the lower total volume collected when compared to liquid L-PRF.Eppendorf of liquid PRF(200g for 8 minutes)produced a significant increase in both the number and concentration of platelets and leukocytes when compared to the i-PRF protocol(~2 fold increases).Furthermore,solid-PRF membranes produced via Eppendorf centrifugation produced the greatest number and concentration of leukocytes and platelets(up to 3.5 fold higher when compared to A-PRF)more evenly distributed throughout the PRF clots when compared to either centrifuges.Conclusions: The present study revealed for the first time the precise location of cells following centrifugation at various protocols utilizing this novel quantification method.Part 3: New Method for Harvesting Concentrated Platelet Rich Fibrin(C-PRF)Objectives: Recently our group proposed a novel method to investigate cell separation following centrifugation of platelet rich fibrin(PRF)using 1m L sequential layers.Owing to these novel findings,it was revealed that cell accumulation following a leukocyte and platelet rich fibrin(L-PRF)protocol(2700 RPM for 12 minutes;~700g)demonstrated a massive accumulation of cells directly above the red corpuscle layer(buffy coat)whereas injectable-PRF(i-PRF)protocols(800 RPM for 3 minutes;~60g)revealed only a slight increase in platelet/leukocyte concentrations following centrifugation.The purpose of this study was to develop a novel harvesting technique for liquid-PRF with highly-concentrated formulations of platelets/leukocytes.Materials and Methods: Standard high g-force PRF and low g-force i-PRF protocols were utilized to separate blood layers.Above each of the red blood corpuscle layers,sequential layers of 100 u L were harvested(12 layers total;ie 1.2 m L which represents the total i-PRF volume)and 3 layers(3x100u L)harvested from the red blood cell layer were collected.Each layer was then sent for complete blood count(CBC analysis)and investigated for cell numbers.Results: The i-PRF protocol revealed typically a 2-3 fold increase in platelet and l.5-fold increase in leukocyte concentration throughout the 1-1.2m L pasma layer when compared to whole blood.While relatively no cells were found in the first 4m L layer of L-PRF,a massive accumulation of platelets and leukocytes were found within the buffy coat with extremely concentrated cells 0.3-0.5m L(up to 80-fold increases)above the red corpuscle layer.We therefore proposed harvesting this 0.3m L and 0.5m L layer directly above the red blood cell corpuscle as an injectable concentrated-PRF(C-PRF).While in general,i-PRF was able to increase platelet numbers by ~250%,we demonstrated that a general 1000-1500% increase in platelet numbers could easily be achieved by harvesting 0.3-0.5m L of C-PRF(total platelet concentrations of >2000-3000x109 cells/L).Conclusions: While conventional i-PRF protocols demonstrate an ability to increase platelet yield by 2-3 fold and leukocytes within a 50% increase,we demonstrate convincingly an ability to concentrate platelet and leukocytes over 10 fold by harvesting specifically by collecting 0.3-0.5m L of C-PRF within the buffy coat.Clinical Relevance: This study revealed a more effective way to concentrate platelets and leukocyte formulations of liquid PRF utilizing a novel 0.3-0.5m L layer of C-PRF.